System and method for the optical measurement of stability and aggregation of particles

ABSTRACT

The present invention relates to a method for the optical measurement of at least the stability and the aggregation of particles in a liquid sample which is located in a sample container, wherein the method comprises the following steps: irradiating the sample with light of at least one first wavelength in order to fluorescently excite the particles, irradiating the sample with light of at least one second wavelength in order to examine the scattering of the particles, measuring the fluorescence light which is emitted by the sample; and measuring the extinction light at the second wavelength, wherein the irradiated light of the second wavelength runs through the sample container, is reflected back, runs again through the sample container in the opposite direction and exits as extinction light, wherein the stability is determined based on the measured fluorescence light and the aggregation is measured based on the measured extinction light. The invention further relates to a corresponding apparatus.

CROSS-REFERENCE TO RELATED APPLICATION

This Application is continuation of U.S. application Ser. No.16/750,902, filed on Jan. 23, 2020, which published as US 2020/0158614A1 on May 21, 2020, which is a continuation of U.S. application Ser. No.15/765,019, filed on Mar. 30, 2018, which issued as U.S. Pat. No.10,545,081 B2 on Jan. 28, 2020, which is a Section 371 National StageApplication of International Application No. PCT/EP2016/073471, filed 30Sep. 2016 and published as WO 2017/055583 A1 on 6 Apr. 2017, in German,the contents of which are hereby incorporated by reference in theirentireties.

The invention generally relates to an apparatus or a system and a methodfor the optical measurement of the stability of particles. Inparticular, the invention relates to a system and a method by means ofwhich not only the stability of particles but also the aggregation ofparticles can be measured optically. According to the invention,preferably the stability and aggregation of the particles may bemeasured with one single apparatus, preferably simultaneously or almostsimultaneously.

BACKGROUND OF THE INVENTION

Since active agents, like for example antibodies, have been developed soas to be active only in their native form, denatured active agents areoften not effective and have to be avoided. Denaturation means astructural transformation of the biomolecules, for example proteins,which in most cases is connected with a loss of the biological functionof said molecules. Denaturation may be the consequence of eitherphysical or chemical influences. Thus, active agent formulations have tobe developed which prevent the denaturation of drugs, i.e. stabilizethem for example thermally, chemically and/or with respect to time.

Aggregating active agents may lead to ineffectiveness as well.Furthermore, aggregated and/or denatured particles, for exampleaggregated antibodies, may provoke a reaction of the immune system inthe body and thus have to be either avoided in drugs or their percentagein the drug has to be minimized.

Denaturation of particles, for example antibodies, has per se to beavoided since it reduces the effectiveness. The aggregation ofparticles, for example antibodies, has per se to be avoided since itprovokes a reaction of the immune system and may also lead to areduction of effectiveness.

It is often unclear why a particle aggregates and/or denatures: Does aparticle aggregate because it denatures, i.e. because it is not in itsnative form or does it aggregate in its native form and denaturesafterwards? Thus, in order to comprehensively characterize theparticles, it is often not sufficient to analyze merely the aggregationor merely the denaturation separately from each other.

With the inventive system and method the denaturation as well as theaggregation of particles may be measured. In particular, with theinventive system and method, the denaturation as well as the aggregationof particles may be measured virtually simultaneously (substantiallysimultaneously) or simultaneously.

The denaturation of particles is an “intra particle” procedure and maybe measured with the inventive method and system by means of measuringthe intrinsic particle fluorescence (for example tryptophanfluorescence, tyrosine fluorescence). Simultaneously, the aggregation ofthe particles, an “inter particle” procedure which changes the size ofthe particles, may be measured by means of scattering of unabsorbedlight.

Since the scattering of light, for example the static scattering oflight in the case of the Rayleigh scattering, depends on the sixth powerof the size of the particle (radius), it is very suitable to measure thechanges the particle size and thus the aggregation of particles. Saidlight scattering method is known and is used by many apparatuses andmethods. In particular, the devices known from the prior art measure thescattered light of the particles at determined solid angles, i.e. theshare of light which is scattered by a particle in a determined solidangle vis-à-vis the incident light. The larger the particles and thesmaller the wavelength, the larger the intensity of the scattered lightfor a fixed, suitably determined angle becomes (cf. for examplehttp://www.Isinstruments.ch/technology/static_light_scattering_sls/).Such a method is described for example in the application US2014/0234865 A1.

From an increase in the scattered light, for example during increase inthe temperature, these methods may conclude an amendment in size andthus aggregation of the particles. A person skilled in the field oflight scattering procedures knows that it has to be avoided that theexcitation light which is beamed onto particles to be examined gets intothe detection optics. A skilled person will always constructcorresponding apparatuses in such a manner that a direct detection ofsaid excitation light is avoided or the excitation light is blocked,which requires significant technical effort. It is for example describedin patent DE 10 2007 031 244 that reflections are undesired with respectto scattering light measurements and also reflections at glass cuvettesmay lead to problems.

Thus, there is the need for an improved or alternative system or animproved or alternative method for measuring the stability and theaggregation of particles.

SUMMARY OF THE INVENTION

The inventive apparatus and the inventive method are defined by thefeatures of the independent claims. Advantageous embodiments can betaken from the subclaims.

The invention relates to a method for optically measuring or determiningthe stability and/or aggregation of particles in a liquid sample whichis located in a sample container. According to the invention,aggregation may be measured independently of stability; preferably,however, aggregation as well as stability are determined. The inventivemethod comprises at least one of the following steps.

The sample is irradiated with light or a light ray of a firstwavelength, in particular in order to stimulate the particles tofluorescence. The light of the first wavelength is thus a fluorescenceexcitation light. In order to determine the fluorescence of a sample thefluorescent light which is emitted by the sample is measured. Typically,the wavelength of the fluorescent light differs from the firstwavelength of the fluorescent excitation light. Based on the measuredbrightness or intensity of the fluorescent light, information about thestability of the particles may be given. Preferably, the detectormeasures the fluorescence with fluorescent excitation light in awavelength range from 260 nm to 300 nm, further preferably in awavelength range from 270 nm to 290 nm and the fluorescent emissionlight in a wavelength range from 320 nm to 380 nm.

The aggregation of particles is determined by irradiating the samplewith light of a second wavelength, preferably with a first intensity I₀.According to the invention, a first or second wavelength may correspondto an exact wavelength as it is provided for example by a laser.According to the invention, the term first and second wavelength mayalso be a “medium” wavelength or “central” wavelength, i.e. in the senseof a wavelength range. For example, wavelength ranges are emitted by alight source if the light source is not a laser. According to theinvention, preferably LEDs are used which emit light over a small orlarge wavelength range. In order to limit the range of the wavelength, aband-pass filter=“excitation filter” is preferably incorporated into thepath of rays. For example, a band-pass filter may have a band-pass widthbetween 30 nm and 1 nm in order to maintain the desired excitationwavelength range. This is particularly preferred with respect tofluorescence so that light which is emitted by the LED is limited to awavelength range which is not in the wavelength range of thefluorescence emission detection. Furthermore, it is also preferred touse an LED for the extinction measurement. Also in this case a band-passfilter having a suitable band-pass width may be used analogously inorder to limit the wavelength range which is emitted onto the sample toa “second wavelength” (second wavelength range).

The scattering of the particles is preferably determined with the secondwavelength. According to the invention, the extinction light is measuredat the second wavelength wherein the ratio of the irradiated light I₀ ofthe second wavelength, which runs through the sample container, and theemergent light I (intensity I), preferably also at the secondwavelength, describes the extinction. Preferably, the entering radiationI₀ runs through the sample container, is reflected, runs through thesample container substantially opposite to the entering direction andsubsequently exits as light I which is also referred to as extinctionlight in the sense of the present invention. Based on the measuredbrightness or intensity I of the exiting light (extinction light), inparticular in relation to the intensity of the irradiated light I₀,information about the stability of the particles may be obtained.According to the invention, a pure aggregation measurement may beconducted also without the above-identified fluorescence measurement.

Preferably, the second wavelength is chosen such that the particles inthe sample to be examined are not absorbed or only very slightlyabsorbed at said wavelength, preferably less than 10%, further preferredless than 5%, further preferred less than 4%, 3%, 2% or 1%.

Further preferred less than 0.1%. Furthermore, it is preferred that thewavelength is chosen with respect to the absorption behavior of theparticles and not with respect to the complete “sample” or “sampleliquid”, since possibly additions in the sample or sample liquid arepresent which absorb with respect to the liquid chosen. However, sincethe invention examines stability and aggregation of the particles, theabsorption behavior of the remaining components may be assumed to be“constant”.

For example, it is known that proteins absorb light in the range between200 nm to 300 nm because of their peptide bonds (maximum of absorptionat approximately 220 nm) and their amino acids (maximum of absorption atapproximately 280 nm). Thus, according to the invention, preferablylight having a wavelength larger than 300 nm is used (cf. FIG. 16).Preferably, the first and the second wavelengths are different.Alternatively, the first and the second wavelengths may be also equal.

In order to measure fluorescence, at least one first wavelength is used.According to the invention, it is also possible that for thefluorescence measurement a further wavelength is used in addition to thefirst wavelength. Thus, for example a first fluorescence may be excitedat a wavelength of 280 nm and a second fluorescence at a secondfluorescence channel at 632 nm.

Correspondingly, according to the invention, at least one secondwavelength may be used for measuring the extinction. For example,extinction may be measured at two different wavelengths; for example at385 nm and 532 nm. Here, it is for example possible to form and evaluatea ratio of the measured values at both wavelengths, in order to quantifyfor example the Mie scattering.

In other words, according to the invention it is possible to use two ormore fluorescence channels and/or two or more extinction channels fordetermination. Preferred embodiments of the present invention orpreferred feature combinations of the present invention are described inthe following exemplary aspects:

-   -   1a. Method for the optical measurement, in particular of the        stability and/or the aggregation of particles, ligands and/or        particle-ligand complexes in a liquid sample, which is located        in a sample container. Preferably, the sample container is        arranged on a reflecting surface, wherein the sample container        is at least partially in contact with the surface.    -   1b. The method according to any one of the preceding aspects        preferably comprises the step of: irradiating the sample with        light of at least one first wavelength, or at least one first        wavelength range, to fluorescently excite the particles,    -   1c. The method according to any one of the preceding aspects        preferably comprises the step of: irradiating the sample with        light of at least one second wavelength or at least one second        wavelength range to examine the scattering of the particles        and/or a ligand bonding.    -   1d. The method according to any one of the preceding aspects        preferably comprises the step of: measuring the fluorescence        light emitted by the sample.    -   1e. The method according to any one of the preceding aspects        preferably comprises the step of: measuring the extinction light        at the at least one second wavelength or in the at least one        second wavelength range.    -   1f. The method according to the preceding aspect, wherein the        irradiated light of the at least one second wavelength or the at        least one second wavelength range is irradiated into the sample        container such that it at least partially runs through the        sample container, is reflected back by the surface, runs again        at least partially through the sample container in substantially        opposite direction and exits as extinction light.    -   1g. The method according to any one of the preceding aspects        preferably comprises the step of determining the stability on        the basis of the measured fluorescence light and/or the        aggregation and/or the ligand bonding on the basis of the        measured extinction light.    -   2. The method according to aspect 1, wherein the fluorescence        light and the extinction light are measured with a common        optical system.    -   3. The method according to any one of aspects 1 or 2, wherein        the irradiation of the sample with the first and second        wavelength is not conducted simultaneously; or the irradiation        with the second wavelength is conducted continuously, whereas        the irradiation with the first wavelength is conducted        intermittently, preferably periodically.    -   4. The method according to any one of the preceding aspects,        wherein the fluorescence light and the extinction light are        measured sequentially, almost simultaneously and/or emitted.        Almost simultaneously is preferably within 4 ms, 2 ms or 1 ms at        most. For example, only the light of the first wavelength may be        switched on for 1 ms and subsequently the light of the second        wavelength may be switched on for 1 ms so that almost        simultaneously means within 2 ms. According to the invention,        simultaneously is preferred. A simultaneous measurement may for        example be achieved with the configuration of FIG. 8. The        simultaneous measurement has the particular advantage of higher        efficiency or performance. Thus, with the configuration of FIG.        8, for example, when measuring aggregation, a 5 times higher        performance is possible than with FIG. 7.    -   5. The method according to any one of the preceding aspects,        wherein the extinction light and the fluorescence light are        measured by a common detector (cf. for example FIG. 6); the        extinction light is measured by a first detector and/or a second        detector, and fluorescence light of a first fluorescence        wavelength is measured by a first detector and fluorescence        light of a second fluorescence wavelength is measured by a        second detector (51) (cf. for example FIG. 7); or the extinction        light is measured by a first detector, fluorescence light of a        first fluorescence wavelength is measured by a second detector        and fluorescence light of a second fluorescence wavelength is        measured by a third detector (cf. for example FIG. 8).    -   6. The method according to any one of the preceding aspects        wherein the sample container is a capillary.    -   7. The method according to any one of the preceding aspects,        wherein the sample container is tempered (heated or cooled),        preferably rests on a tempering element (heating or cooling        element) and is tempered by contact, wherein the tempering        element preferably comprises a reflecting surface and preferably        reflects back the irradiated light of the second wavelength,        again runs through the sample container (30) in opposite        direction and exits as extinction light.    -   8. The method according to aspect 7, wherein the tempering        element is made of a material which has little autofluorescence.        Preferably, the material has an autofluorescence of less than        5%, 3%, further preferred of less than 1%, further preferred        less than 0.5% of the maximum fluorescence signal. In other        words, for example if an excitation LED with maximum output        emitted light and a fluorescence detector measured a maximum of        100 signals (before it saturates), merely a signal strength of 1        may be traced back to the autofluorescent material; this would        be 1%. It is further advantageous when the material has a high        reflectivity in the wavelength range of the second wavelength,        preferably >30%, preferably >40%, further preferably >50%.        Preferably, the material contains silicon or consists of pure        silicon.        -   According to a further preferred embodiment, the surface has            at least one recess, for example the form of a furrow,            groove, or micro groove which extends over at least a region            of the surface of the tempering element on which the            capillary rests during the measurement. Preferably, the            capillary is in direct contact with the surface of the            tempering element during the measurement, whereas the            capillary lies above the groove due to the depth of the            groove and is not in direct contact with the bottom of the            groove. Preferably, the groove is between 1-10 mm, more            preferred between 2-8 mm, further preferred between 3-7 mm,            further preferred approximately 3 mm wide, wherein the            inventive back reflection of the light is preferably            produced or measured in the region of the capillaries which            lies above the groove. Preferably, the groove has a depth of            approximately 10-30 μm. In particular, the groove has a            depth of more than half of the coherence length of the used            light in order to further suppress interference effects in            the back scattering. Hence, preferably used LED light            sources have coherence wavelengths in the range of            approximately 15 μm so that a depth of the groove of >7.5 μm            is preferred.        -   In order to guarantee an efficient back reflection of the            light from the bottom of the groove, the groove is            preferably etched. Preferably, the groove or the bottom of            the groove has an average roughness which is preferably in            the nanometer range, for example ±5 nm, preferably ±1 nm.            According to a preferred embodiment, the groove may extend            over a substantial part of the surface so that for example            several capillaries may be arranged above the groove.            According to a further preferred embodiment, the groove does            not extend until the edge of the surface so that the silicon            has a constant thickness around the groove and may thus be            easier processed, for example by cutting or sawing.    -   9. The method according to any one of the preceding claims,        wherein the sample container is shifted during a measurement        period relatively to the irradiated light of the first and/or        second wavelength and/or to the detector, is preferably run back        and forth several times (continuously) and further preferably a        plurality of sample containers or a plurality of capillaries are        scanned by said relative movement.    -   10. The method according to aspect 9, wherein a fluorescence        value is determined by integrating the intensity of the        fluorescence light via the shifting and/or an extinction value        is determined by integrating the intensity of the extinction        light via the shifting.    -   11. The method according to any one of the preceding aspects,        wherein during a measuring period, in order to determine the        thermal stability, the temperature of the samples is changed,        preferably increased; in order to determine chemical stability,        the concentration of denaturants in different liquid samples is        chosen differently; and/or in order to determine stability in        terms of time, the sample is kept at a substantially constant        temperature for a time period of more than one hour.    -   12. The method according to aspect 11, wherein during a        measuring period a plurality of sample containers and/or the        optical system are continuously run back and forth several times        and the measurements of the fluorescence light and/or the        extinction light are conducted during the movement.    -   13. The method according to any one of the preceding aspects,        wherein the second wavelength is chosen such that less than 1%,        0.1%, 0.05%, preferably less than 0.1%, is absorbed by the        sample or the particles in the sample so that the measurement of        the extinction light is a direct rate for the scattering of the        light of the second wavelength.    -   14. The method according to any one of the preceding claims,        wherein the light of the first wavelength and the light of the        second wavelength are united to a collinear ray which is        irradiated into the sample container.    -   15. The method according to any one of the preceding claims,        wherein the extinction light of the second wavelength, which is        reflected back and leaves the sample container in the opposite        direction to the irradiation direction, deviates from the        irradiation direction 5° at most, preferably less than 2°,        further preferred less than 1°.    -   16.a An apparatus for the optical measurement, in particular of        the stability and/or the aggregation of particles and/or ligands        and/or particle ligand complexes in a liquid sample, which is        located in a sample container, in particular according to any        one of the preceding aspects.    -   16.b The apparatus according to any one of the preceding        aspects, wherein the apparatus comprises: at least one first        light source for irradiating light of at least a first        wavelength into the sample container, in particular in order to        fluorescently excite the particles to be examined    -   16.c The apparatus according to any one of the preceding        aspects, wherein the apparatus comprises at least one second        light source for irradiating light of at least one second        wavelength into the sample container, in order to measure the        scattering or aggregation of the particles and/or ligand        bonding.    -   16.d The apparatus according to any one of the preceding        aspects, wherein the apparatus comprises at least a first        detector for measuring the excited fluorescence light which is        radiated from the sample.    -   16.e The apparatus according to any one of the preceding        aspects, wherein the apparatus comprises at least one second        detector for measuring extinction light at the at least one        second wavelength wherein the irradiated light of the second        wavelength runs through the sample container, is reflected back,        runs again through the sample container in the opposite        direction and exits as extinction light.    -   16.f The apparatus according to any one of the preceding        aspects, wherein the apparatus comprises an evaluation means        which determines the stability of the particles based on the        measured fluorescence light and which determines the aggregation        of the particles and/or ligand bonding based on the measured        extinction light. Preferably, the apparatus comprises a first        and/or second bandpass filter to narrow down the emitted light        of the first and second light sources to the first and second        wavelength, respectively. Preferably, a bandpass filter has a        bandpass width of 10 nm, 20 nm or 30 nm.        -   Preferably, the apparatus has a tempering element with a            reflecting surface at which the irradiated light of the            second wavelength is reflected back. For example, it came            apparent that silicon is particularly preferred since it has            a preferred reflection behaviour and is suitable for the            tempering via contact. Further, it is preferred that the            apparatus is suitable to arrange at least one sample            container on the surface for measurement purposes. For            example, several sample containers in the form of separately            arranged capillaries or by means of a carrier which            comprises a plurality of capillaries may be arranged on the            surface. Preferably, at least one groove is configured in            the surface of the tempering element, wherein the sample            container may be arranged above the groove in such a way            that the irradiated light of the second wavelength is            preferably reflected back at least from the bottom of the            groove. For example, a groove having a width of between 1-10            mm and a depth of more than half of the coherence length of            the light of the second wavelength may be configured.    -   17. Use of an apparatus according to aspect 16 for conducting a        method according to any one of aspects 1 to 15.

The inventive method or inventive system has a completely differentapproach compared to conventional light scattering measurements.According to the invention, preferably the light which is not scatteredis measured. Furthermore, preferably light having a wavelength which isnot absorbed by the particles is used. That means the measured signaldecreases when the scattering increases due to an increase in the sizeof the particles. Said inventive measurement technique is preferablycombined with specific fluorescence optics which preferably enable afaster, more precise and more rugged simultaneous detection ofaggregation (by extinction) and detection of denaturation or unfoldingof proteins (by fluorescence) in high output.

The inventive method is preferably configured in a manner that theexcitation light for the aggregation measurement runs through the samplecontainer twice and is reflected back to the detector (cf. FIG. 1).According to the invention, it is also possible that the excitationlight for the aggregation measurement runs through the sample containeronly once and subsequently the one way transmission is measured. Directtransmission as well as transmission after reflection mean that a“residuary portion” of the excitation light is measured, i.e., exactlywhat should have been avoided in the known methods.

According to the invention, in principle extinction is measured (cf. forexample https://de.wikipedia.org/wiki/Extiktion (Optik)). In optics, theextinction or optical density is the perceptional logarithmicallyformulated opacity O and thus a rate for the dilution of a radiation(for example light) after a medium has been run through. I₀ being theincoming radiation and I being the exiting radiation, extinction Edescribes the transmission degree ti as logarithmic value:

$E_{\lambda} = {{{- \log_{10}}\frac{I}{I_{0}}} = {{\log_{10}\frac{I_{0}}{I}} = {{\log_{10}\frac{1}{\tau_{\lambda}}} = {\log_{10}O_{\lambda}}}}}$

Generally, the processes of absorption, scattering, deflection andreflection are involved in dilution/extinction. Since, according to theinvention, preferably wavelengths are used which are not absorbed by theparticles to be examined (for example biomolecules) and otherinfluencing variables as reflection and deflection are preferably keptconstant, according to the invention substantially the dilution based onthe pure scattering is measured.

This approach is particularly advantageous since said “scattering”measurement principle may well be integrated into a (single) opticalsystem for measuring the intrinsic particle fluorescence. Thus, withonly one optical system, the denaturation of the particles in the nmscale as well as their aggregation in the nm-μm scale may be detected ormeasured. Both measurements may be carried out sequentially, shortlyafter each other or even simultaneously, depending on the configuration.

According to the invention, the samples to be examined are examinedpreferably in capillaries, which additionally has the preferredadvantage that the capillaries may be quickly brought in the desiredmeasurement position, which makes it possible to analyze a plurality ofsamples simultaneously. Furthermore, this guarantees a high density ofdata points which makes it possible to precisely determine and evaluateeven small signal changes, which is up to now not guaranteed by existingmethods.

According to the invention, a plurality of capillaries may be laiddirectly onto an array element of the measurement device. According to afurther embodiment, a plurality of capillaries may also be arranged on aseparate array, which enables a semi-automatic or automatic fillingand/or measurement.

Applicant of the present invention, NanoTemper Technologies GmbH,develops and sells measurement devices by means of which liquids withina capillary are optically examined. It is further known that anindividual capillary is taken by hand, immerged into a liquid andsubsequently positioned separately on an array and then pushed into themeasurement device.

Said method for filling separate capillaries is shown for example in avideo of NanoTemper Technologies GmbH, which is published underhttp://www.youtube.com/watch?v=rCot5Nfi_Og. The individual filling isadvantageous for certain individual samples, however, for larger amountsof samples said method needs many handling steps which cannot readily beautomated.

In the application EP 2 572 787, which has been filed by the sameApplicant as the present invention, capillaries are described which arekept to an array by means of magnetic forces. This, i.a., enables aneasier and/or more exact positioning of the individual capillaries onthe array. In other words, the individual filling of the individualcapillaries is further preferred, however, the subsequent step issupported by the magnetic forces.

Finally, in the application EP 2 848 310 a separate array forcapillaries is described, which enables also a semi-automatic orautomatic filling and/or measurement. In particular, said arrays mayalso be used for the inventive method, which has the additionaladvantage that the plurality of capillaries may not only be efficientlyfilled but also scanned very fast.

In the following, some terms are defined as to how they should beunderstood in the context of the present application.

Particles

Particles in the context of the present application are, without beinglimited thereto, preferably: active agents, biomolecules in general, forexample proteins, antibodies, membrane proteins, membrane receptors,peptides, nucleotides, DNA, RNA, enzymes; molecule fragments, “smallmolecules”, sugars, organic bonds, anorganic bonds; vesicles, viruses,bacteria, cells, micelles, liposomes, tissue samples, tissue cuts,membrane preparations, microbeads and/or nanoparticles.

Fluorescence Measurement

The particle, preferably protein, may be denatured chemically orthermally and internal structural changes may be measured by intrinsicfluorescence, for example tryptophan fluorescence, tyrosinefluorescence, phenylalanine fluorescence, preferably tryptophanfluorescence in the case of proteins. Here, the structural/internalchanges of the particle may be detected by means of changes in theintensity of the fluorescence or shifting of fluorescence maxima orchanges in the fluorescence lifetime etc. The so-called melting point ofthe particle to be examined, for example protein, may also be determinedin this way. The melting point is defined as the state in which theparticle to be examined, for example protein, is half-folded (forexample protein: in native conformation) and half-unfolded (for exampleprotein: unstructured, denatured shape). In this context, the change inthe fluorescence intensity may be determined for example depending onthe temperature or the addition of a denaturant or co-factor/ligandand/or a temporal course may be recorded.

If proteins are examined, for example the tryptophan fluorescence at awavelength of 330 nm+/−10 nm and 350 nm+/−10 nm may be measuredsimultaneously but spectrally separated. The quotient of thefluorescence intensity at 350 nm and the fluorescence intensity at 330nm (F350/F330) is a preferred measured value since it depends on theinternal structure or conformation changes of the particle. Thefluorescence emission maximum of tryptophan, for example, shifts fromshort wavelengths (for example 330 nm+/−10 nm) to long wavelengths (forexample 350 nm+/−10 nm) when the tryptophan gets out of its hydrophobicenvironment, for example inside a protein, due to unfolding of aprotein, and into a hydrophilic environment, for example water. Forexample, the melting point may be determined from the maximum of thefirst derivation of the F350/F330 curve.

Extinction/Scattering Measurement

Particles in solutions are able to scatter irradiated light. Scatteringin physics generally means the deflection of an object by interactionwith a locally different object (scattering center). Scattering of lightat the particles is thus the deflection of the irradiated (excitation)light by interaction with a particle to be examined. The scatteringangle θ is defined as the angle about which the scattered light isdeflected. According to the invention, scattering is meant when thelight is indeed deflected, preferably about more than 1°, preferablymore than 2°, 3° 4° and preferably less than 179°, 178°, 177°, 176°,measured from the course of the rays of the irradiated (excitation)light.

A distinction is made between different kinds of scattering, as forexample Rayleigh scattering (particle dimensions ˜ 1/10 of the lightwavelength, i.e. particle dimensions which are small compared to thelight wavelength) and Mie scattering (particle dimensions in the rangeof the light wavelength and larger). The extent of the scattering in asolution depends on the dimension and number of particles. Since thescattering intensity of the Rayleigh scattering with the inverse 4^(th)power depends on the wavelength, it is more distinct in short wavelengthranges, for example 300-400 nm, than in long wavelength ranges. Theextent of the scattering may be quantified by extinction measurement, bycomparing the intensity of the irradiated light to the intensity of thetransmitted light. The difference corresponds to the amount of scatteredlight and thus serves as rate for the formation of particles oraggregation of particles.

In the case of biomolecules, for example proteins, the detection ofextinction at a wavelength higher than 300 nm is advantageous. Inparticular, the detection of the extinction between 300 and 400 nm isadvantageous and at approximately 385 nm particularly advantageous sincehere substantially no light is absorbed (absorption maximum of proteinsis at approx. 280 nm), however, the scattering due to the dependency ofthe wavelength of the Rayleigh scattering is very high. For example, theuse of light at 385 nm is advantageous since appropriate LEDs at themarket are more efficient at said wavelength than LEDs having asignificantly shorter wavelength. In particular, a strong light outputis advantageous in order to detect many photons. Hence, the extinctionmeasurement is often limited by photon noise. The signal-noise ratio ofthe photon noise follows a Poisson distribution, i.e. it improves withthe root (number of photons).

Additionally, the above-mentioned selection of the wavelength for theextinction has further advantages. Since the light is not absorbed bythe particles, the particles are not destroyed so that a “strong” lightoutput in this wavelength range may be used. The light output of the LEDfor the extinction measurement is preferably in the range of higher than100 μW, preferably in the range of higher than 1 mW. Preferably, thelight output of the LED for the extinction measurement is in the rangeof 0.1 μW to 5 mW.

For measuring the extinction, little noise of the signal and a littledrift of the excitation light source are advantageous. LEDs may beoperated very stably and noise-reduced with suitable LED controllers andare thus advantageous for extinction measurements.

Since biomolecules, for example proteins, are significantly smaller thanthe advantageous wavelength ranges, Rayleigh scattering can be assumed.Since the Rayleigh scattering is dependent on the 6^(th) power of theparticle diameter, changes in the size of the particle, for example dueto aggregation of the particles, lead to a significant change in thescattering. Since the scattering at particles occurs in all spatialdirections, it is proposed, according to the invention, to quantify thescattering or a degree of the scattering via the extinction since thus aquantification of the complete scattering is possible without beingdependent on the scattering angle, contrary to conventional lightscattering measurements in which scattered light is detected only in asmall angular range. Furthermore, extinction measurements are lesssensitive to measurement artifacts, for example reflections at boundarysurfaces and contaminations as for example dust particles.

Preferred wavelengths or wavelengths ranges for extinction measurementscan be derived for example from FIG. 16, in which an absorption spectrumfor proteins is shown. For example, it can be derived from said spectrumthat wavelengths which are larger than 280 nm, preferably larger than300 nm, are particularly preferred.

Sample Containers

According to the invention, samples are examined which are in containersor sample containers in the form of liquids or fluids. In principle, theinventive method is not restricted to a certain kind and shape of samplecontainers. However, preferably capillaries are used as samplecontainers, which has several advantages. For example, the use of thincapillaries leads to a reduced waste of material due to the smallvolume. Furthermore, thin capillaries have high capillary forces inorder to suck in the liquid passively, only by their capillary forces.Even highly viscous liquids may be sucked into the capillaries by thecapillary forces. It is for example also possible to turn the sample tobe sucked in upside down so that also gravitational forces act in thedirection of the capillary forces and thus support the filling. The useof one-way capillaries avoids the cross-contamination between theindividual samples. Thin capillary means that the optical path lengththrough the capillary is small. This is advantageous for measuring alsovery highly concentrated solutions (high concentration of particles).According to the present invention, for example individual capillariesmay be used or capillaries in arrays. Thus, it is for example possibleto place an array with several capillaries on the reflecting surface,wherein the several capillaries preferably are in contact with thesurface without having to remove the capillaries from the array. Inother words, the capillaries may be tempered by contact with the surfacevia contact tempering, while the capillaries remain in the array.

Preferred arrays with capillaries are described for example in EP 2 848310, which is incorporated herein by reference. In particular, EP 2 848310 relates to an array for several capillaries which enablessimultaneous filling of several capillaries of a microwell plate.Furthermore, EP 2 848 310 also relates to an apparatus and a method forfilling, transporting and measuring liquids having volumes in the themicroliter range. According to a preferred embodiment, 24 capillariesmay be arranged in one array.

The liquid sample is preferably in a static, i.e. non-fluent stateduring the measurement within the capillaries. Preferably, during themeasurement there are no flows within the capillary which go beyond thenatural temperature movement and/or possible movements due toevaporation in the liquid.

The capillaries may be made of glass and/or a polymer and/or at leastone of the elements of borosilicate glass, borosilicate 3.3 glass (forexample DURAN-glass), quartz glass like suprasil, infrasil, syntheticfused silica, soda-lime glass, Bk-7, ASTM Type 1 Class A glass, ASTMType 1 Class B glass. The polymers may comprise: PTFE, PMMA, Zeonor™,Zeonex™, Teflon AF, PC, PE, PET, PPS, PVDF, PFA, FEP, and/or acrylicglass.

In particular, it is preferred that at least one range of thecapillaries is transparent for light having a wavelength of 200 nm to1000 nm, preferably from 250 nm to 900 nm. Particularly preferred, butnot limited thereto, said range of the capillary is also transparent forlight having the following wavelength ranges: from 940 nm to 1040 nm(preferably 980 nm+/−10 nm), from 1150 nm to 1210 nm, from 1280 nm to1600 nm (preferably 1450 nm+/−20 nm and/or 1480 nm+/−20 nm and/or 1550nm+/−20 nm), from 1900 nm to 2000 nm (preferably 1930 nm+/−20 nm). Theskilled person understands that the transparent range(s) may also extendover the complete capillary. In other words, the capillaries may betransparent and are preferably made integrally of one of theabove-mentioned materials.

Preferably, the used capillaries have an inner diameter of 0.1 mm to 0.8mm, preferably 0.2 mm to 0.6 mm, further preferably 0.5 mm. The outerdiameter of preferred capillaries is preferably between 0.2 mm to 1.0mm, preferably 0.3 mm to 0.65 mm.

The geometry of the capillaries is not limited to a certain shape.Preferably, tube-like capillaries having a round cross-section or anoval cross-section are used. However, it is also possible to usecapillaries having a different cross-section, for example, triangular,quadrangular, pentagonal or polygonal. Furthermore, capillaries may beused which have a diameter and/or cross-section which is not constant orconstant over the length of the capillaries.

Silicon Surface

According to the invention, the sample containers are on a siliconsurface. Preferably, capillaries which are arranged above a siliconsurface are used as sample containers. The silicon surface preferablyserves as reflecting surface or surface for the reflection of theexcitation light. Furthermore, according to the invention, the samplecontainers or capillaries may be brought into direct contact with thesilicon surface so that a direct contact heat exchange between samplecontainer/capillary and silicon is achieved. Silicon has some propertieswhich are particularly advantageous for the present invention.

Silicon does not have autofluorescence in the preferred wavelengthrange. Silicon has a high reflection in the wavelength range which ispreferred according to the invention (cf. for example FIG. 9).Furthermore, silicon has a high thermal conductivity which isparticularly advantageous for the fast and homogenous tempering of aplurality of capillaries. Said three properties are particularlyadvantageous for the inventive apparatus and the inventive method.

Further advantages of silicon are for example its chemical resistanceand that it is easily available and easy to produce, which furthermoremakes it possible to form very smooth or very precise shapes/surfaces.

However, the invention is not limited to the use of silicon. Thus, othermaterials may be used for the reflecting surface and/or temperinginstead of silicon. Generally, materials are suitable which have lowautofluorescence or no autofluorescence in the preferred wavelengthmeasurement range and preferably simultaneously show reflection of theirradiated light, for example >10% reflection. According to theinvention, for example also a quartz layer or a quartz plate may be usedwhich is preferably provided with a reflecting coating, for example aninterferometric coating.

The present invention offers several advantages with respect toefficiency, speed and costs for conducting a plurality of measurements.The combination of thin capillaries which preferably rest on silicon andwhich are preferably continuously shifted relative to a single opticalsystem together with the measurement of the intrinsic fluorescence(fluorescence, phosphorescence, luminescence) and the measurement of thescattering (extinction) is preferred and particularly advantageous. Inparticular, silicon is a preferred material due to the combination ofthe very good heat conducting properties and the property to reflectlight of the used wavelength. The fast and precise detection ofextinction by running a plurality of samples without resting onindividual capillaries also drastically increases the measurement speedand data point density compared to conventional methods.

The inventive method and the inventive apparatus fundamentally differfrom the prior art. In particular, according to the invention,components are combined in a way which a person skilled in the art oflight scattering would not use based on the teaching of the prior art.Furthermore, the inventive measurement also differs from known methods aperson skilled in the art of absorption measurements would conduct. Inprinciple, there are commonalities between the optical system for anabsorption measurement and an inventive apparatus. According to theinvention, however, the wavelength for the extinction measurement isspecifically directed such that it is not absorbed by the sample. Thus,according to the invention, a measurement of the scattering of the lightdependent on the dimension may be achieved.

For example, from the prior art an apparatus is known which is soldunder the name UNit™ by unCHAINED LABS. In said apparatus, themicrocuvettes have to be controlled individually and for the measurementthe microcuvettes have to be arranged exactly with respect to themeasurement optics. According to the invention, the capillariespermanently/continuously move without stopping for the measurement; thecapillaries are “completely scanned”. Thus, according to the invention,a more robust, more efficient configuration and especially a much higherdata density is achieved.

Due to the recording of complete spectra according to the prior art, themeasurement time per controlled microcuvette takes several seconds.Thus, the measurement of for example 48 samples at one temperaturealready takes several minutes. A heating rate with the usual 1° C./minthus leads to a small data point density. According to the invention, itis already sufficient that merely two discrete wavelengths are detected(extinction and fluorescence), wherein the capillaries may be exposed tolight for <50 ms each during passing by so that, according to theinvention, a data point density which is by ranges higher is achieved.

In the prior art a static light scattering measured as usual, i.e. theexcitation light is blocked such that indeed only a part of the lightwhich is scattered is measured. According to the invention, however, thetransmitted part or the reflected part is measured, i.e. the light orpart of the irradiated light which is not scattered.

Furthermore, the measurement of the light scattering from the prior artneeds an exact aiming at the samples, which requires extensiveadjustment and regular maintenance. Furthermore, this leads to a poorrepeatability since even small errors in aiming at separate samples maylead to fluctuations in the light scattering signal.

According to the invention, the extinction light is measured after thelight has twice run through the capillary due to reflection. In the caseof high concentration of the particles and a long path length (forexample 1 cm), no light would return through the capillary. Thus,according to the invention, thin capillaries having an inner diameter offor example 0.5 mm are preferred. The solutions/methods/apparatuseswhich are presently available have problems with handling and measuringhighly concentrated solutions, for example highly concentratedantibodies (for example with 150 mg/ml antibodies in aqueous solution).On the one hand since they cannot fill highly viscous liquids into thesample chambers used, on the other hand since their optical path lengthsare too long. Said highly concentrated solutions are, however, veryinteresting for the pharmaceutical industry, in particular theformulation measurement.

The use of thin capillaries allows a large dynamic measurement rangesince the measurements are highly sensitive (little/no autofluorescenceof the capillary material and the silicon in the case of fluorescencedetection, high transmission of the thin-walled capillaries and goodreflection properties/homogeneity of the silicon in the case ofextinction detection), and at the same time allow the measurement ofhighly concentrated solutions (thin optical layer thickness advantageouswith respect to extinction measurements of highly concentratedsolutions). The inventive method is robust vis-à-vis inner filtereffects since each individual sample is referred to itself. Thus, largeranges of material amount concentration, for example from 50 mg/mlprotein to 5 μg/ml protein, may be analyzed in one single measurement.

Property: Continuous Running Back and Forth of the Capillaries Under theOptics:

The preferred continuous relative shifting of the capillaries to theoptics during a measurement (cf. FIGS. 3 and 4) has further advantageswith regard to efficiency and precision of measurement. According to theinvention, a plurality of samples may be measured parallel, for exampleby tempering all samples simultaneously to the same temperature. Theinventive method does not require a long residence time on theindividual capillaries; a directed driving to a specific measurementpoint on the capillary is not needed either, due to which the method isvery robust and very fast.

According to the invention, furthermore, the symmetry of the capillarymay be utilized since the measurement is preferably conductedperpendicular to the longitudinal axis of the capillary. Furthermore,round or cylindrical capillaries (round cross-section) are advantageoussince such capillaries cannot only be manufactured cheaply but alsohaving a good quality and with high preciseness.

Due to the inventive fast scan procedure very high data point densitiesmay be achieved, which has advantageous effects on the data evaluation.In the following, some of said advantages are calculated by means of anexample.

For example, 48 individual capillaries having an inner diameter of 0.5mm and an outer diameter of 0.65 mm are arranged horizontally on thetempered silicon in a distance of 2.25 mm (center of the capillary tocenter of the capillary). Said complete tempering body with thecapillaries is continuously run back and forth under a fixedly mountedoptical system, for example by means of a linear axis, which is operatedfor example by a step motor.

For example, the tempering body and thus the capillaries are scannedunder the optics with a speed of for example 45 mm/s. At this speed, all48 capillaries are started with a distance of 2.25 mm withinapproximately 3 seconds. In particular, by running back and forth, eachcapillary is measured every 3 seconds on average (“on average” since forexample the outermost capillaries are measured twice practicallyinstantaneously by reversing the driving direction and it thus takes 3seconds (driving back)+3 seconds (returning)=6 seconds until thecapillary is again exactly under the optics).

In an exemplary configuration the temperature of the tempering body ismeasured and thus the temperature of the capillaries is continuouslyincreased by a rate of 1° C. per minute during the continuous runningback and forth. Thus, with the temperature ramp of 1° C. per minute, adata density of 20 measurement points per capillary and per minute isachieved, which corresponds to a temperature definition of 0.05° C. onaverage. If the increasing speed of the temperature of 1° C. per minuteis halved to 0.5° C. per minute with a constant number of capillariesand constant running speed, the temperature definition of 0.05° C.doubles (case 1° C. per minute) to 0.025° C. (case 0.5° C. per minute).

Since it is scanned, i.e. measured continuously over the completediameter of the capillaries, the inventive method is more robustvis-à-vis local contaminations (for example dust particles, air bubbles,in particular contaminations which are smaller than the diameter of thecapillary) than conventional methods from the prior art, according towhich it is measured only at one single point of the capillary (cf. forexample UNit™ by unCHAINED LABS). In particular, in the prior artalready little local contaminations may lead to a measurement artifactand to a rejection of the measurement.

A further advantageous aspect of the inventive scanning of thecapillaries is that different layer thicknesses are measured practicallyautomatically due to the scanning of the round capillaries with the“measuring beam”. Thus, for example FIG. 3 shows that the maximum samplethickness/layer thickness is in the center of the capillary. A smallerlayer thickness of the sample liquid is symmetrically at the edges. Thisis for example advantageous for very highly concentrated solutions inthe case of which the scattering is so high that in the center of thecapillary (greatest layer thickness) no signal gets through (thecomplete light is scattered). However, if no signal gets through, nochanges in the signal can be measured. Since, according to theinvention, it is scanned over the complete diameter of the capillaries,measured values >0 are achieved in the edge regions in which thereflected ray of light had to cover a shorter distance through thecapillary. This is a practical advantage since thus a higherconcentration range of samples may be measured in the solution.

Optics for Measuring the Fluorescence and Extinction/Scattering

A further advantage of the present invention can be seen in the opticsfor measuring the fluorescence and extinction, which is constructed in asimpler manner Advantageously, a common optical system may be used forboth measurements. The advantages of the inventive (common) optics arefor example in the fields of adjustment, positioning, materialconsumption, compared to the separate optics from the prior art. Inaddition, the inventive common optical system also saves space.

According to the invention, the fluorescence measurement and theextinction measurement may be conducted subsequently, almostsimultaneously or simultaneously. Simultaneous measurement means: intraparticle (=intramolecular) processes by means of fluorescence may beconducted simultaneously with the measurement of the particles' changein size (=intermolecular) by means of “scattering”/extinction. Thisleads to a direct correlation of both processes. In this way it can berecognized whether denaturation (fluorescence) and aggregation(extinction) start simultaneously or at the same temperature or whetherone process starts before the other. This also leads to a more robustmeasurement.

The fact that the measurements are conducted simultaneously also leadsto a higher or high data density: twice as much data may be measured pertime unit as if extinction and fluorescence are measured separately fromeach other. Thus, the measurement is more precise and the melting pointof a protein and the temperature at which the aggregation of the proteinbegins may be determined more easily.

The inventive configuration of the extinction measurement (“scattering”measurement) is more robust against contaminations, pollutions, airbubbles on and in the capillary than static scattered lightmeasurements.

Preferred concentrations of material amounts of particles, for exampleproteins like antibodies, enzymes, peptides are between 0.001 and 500mg/ml. Advantageous concentrations are between 0.1 and 100 mg/ml.

The inventive configuration and the inventive method make it possible tosimultaneously measure many different concentrations in one singleexperiment. That means that concentrations which differ for example bythe factor 1000 may be measured simultaneously with one and the samemeasurement setting.

By means of the inventive system and method, measurements of the thermalstability of particles, chemical stability of particles as well asstability of particles with respect to time are possible. In thefollowing, examples for measuring the stability are described in moredetail.

Thermal Stability

When thermal stability is measured, the capillaries with particles inaqueous solution or liquid phase are placed on the tempering body, whichcomprises silicon, are the intrinsic fluorescence and (preferablysimultaneously) the scattering/extinction is preferably continuouslymeasured while the temperature of the capillaries is increased from alow value, for example 15° C. to a high value, for example 95° C. (cf.FIG. 13). For example, temperatures of −20° C. to +130° C. and/orportions thereof may be used as well.

At first, the silicon surface is cleaned with a cloth and absoluteethanol by wiping it several times. Subsequently, at least 10 μl of thesamples to be analyzed are prepared, for example antibody solutions indifferent concentrations of material amount, for example between 5 mg/mland 0.3 mg/ml or different biomolecules or identical biomolecules indifferent buffers. 10 μl of each solution are then filled into thecapillaries by means of capillary forces by immerging the capillariesinto the solutions. Filled capillaries are then transferred to acapillary array and subsequently pressed onto the silicon surface bymeans of a lid. By means of a “discovery scan”, which determines theextinction as well as the fluorescence at 330 and 350 nm emissionwavelength of all capillaries within 3-5 seconds at a temperature of 20°C., the luminous intensities are adapted, which may be carried outmanually or automatically in order to avoid overexposure of thedetectors. Subsequently, the temperature range to be measured, forexample 20° C.-95° C., and the temperature ramp, for example 1° C./minare determined. The latter one may be varied for example between 0.1°C./min and 100° C./min After said parameters have been determined, themeasurement is started and the temperature dependency of the sampleextinction and sample fluorescence is simultaneously measured anddisplayed. After the measurement is finished, the temperature isautomatically set back to the starting value.

The analysis of the thermal unfolding curves is carried out for examplevia the determination of the specific unfolding temperature (thetemperature at which 50% of the particles are unfolded), which may becarried out for example by identification of the inflection points byanalysis of the first or second derivation of the raw data or by othermathematical processes. The analysis of the particle formation byextinction measurement is preferably carried out by detecting thetemperature at which the aggregation starts and by determining themaximum extinction.

For example, the reversibility or irreversibility of the unfolding of aparticle may also be determined by means of a thermal stabilitymeasurement. This may be carried out for example by first increasing thetemperature from 20° C. to 95° C. with a temperature ramp of 1° C. perminute and subsequently decreasing the temperature of 95° C. with thesame or a different temperature ramp to 20° C. If an unfolding isreversible, for example, after the process of heating up and coolingdown, the fluorescence ratio of 350 nm to 330 nm reaches again thestarting level/the same value as it had for said process. With thesimultaneous measurement of the aggregation, according to the invention,it may be discovered whether the aggregation leads to an irreversibilityof the unfolding. For example, in case an antibody has different thermalunfolding processes in the fluorescence signal, for example with meltingtemperatures of 60° C. and 72° C. and in case it simultaneously has anaggregation at 75° C. during the extinction, for example it is possibleto heat to 60° C. in a first experiment and then cool down again andheat to more than 75° C., i.e. beyond the aggregation temperature andthen cool down again in a second experiment. If the first experimentshows a reversible unfolding and the second experiment shows anirreversible unfolding, it can be concluded that the aggregation leadsto an irreversible unfolding or prevents the refolding into the nativestate.

Chemical Stability:

When measuring the chemical stability, particles are mixed in aqueoussolutions with increasing concentrations of denaturants, for examplechaotropic salts like guanidinium hydrochloride or urea and filled intocapillaries and placed on the tempering body. The extent of theunfolding of particles is detected at a defined temperature by one-timerunning the capillaries and detecting the fluorescence (cf. FIG. 12).

Fields of use for chemical unfolding are for example the optimization offormulations of proteins, for example antibodies and enzymes, thethermodynamic characterization of particles as well as the active agentresearch.

Stability with Respect to Time

When measuring the stability with respect to time, particles are filledin aqueous solution into capillaries and the extinction and fluorescenceare measured at constant temperature over a defined time period. Withrespect to measurements >3 hours it is advantageous to seal thecapillary ends with suitable substances, for example liquid plastic,adhesive, wax, putty or mechanically by pressing suitable materials, forexample silicone, rubber, plastic, in order to avoid loss of samplematerial by evaporation. Measuring the stability with respect to time isfor example used for the characterization of particles, in particularproteins and active agents, and for the optimization of the formulationof said particles.

Quality Control

When measurements are carried out for the quality control, particlesolutions are tested with respect to their reproducibility or storageand stress tests are conducted. With respect to the latter one, forexample proteins are exposed to conditions which potentially havenegative influence on their folding, for example increased temperature,intensive shaking/stirring, cycles of freezing and unfreezing. Afterconducting the different procedures, the solutions are filled intocapillaries and the fluorescence as well as the extinction of thesamples is detected either with a single capillary scan or in atemperature ramp, for example with 1° C./min from 20° C. to 95° C. Bycomparing to an untreated reference sample, the share of unfolded andaggregated protein may be detected.

Ligand Bonding

Said measurements are also referred to as “thermal shift assays”. Whenmeasuring the ligand bonding, particles, for example enzymes, forexample kinases, together with ligands, for example fragments ofmolecules, incubated in aqueous solution. If a ligand bonds to aparticle, said ligand bonding may influence the stability, for examplethe thermal stability, of the particle and/or its aggregation behavior.For example, a ligand bonding to a particle may increase or decrease itsmelting temperature, the temperature at which 50% of the particle are innative form and 50% are in denatured form, i.e. stabilize or destabilizethe particle. Said shifting of the melting temperature of theparticle-ligand complex vis-à-vis the particle without ligand may bemeasured as “delta T” and thus the bonding of the ligand to the particlemay be detected. The inventive method and apparatuses make it possibleto reliably and reproducibly detect and quantify even the smallestshifting in the melting temperature of delta T>=0.2° C. Differentligands may then be assorted and selected for example by means of theshifting in their melting temperature delta T. With respect toapplications as for example crystallography of proteins, ligands aresearched for which shift the melting temperature of the particle toparticularly high melting temperatures when bonding and thus stabilizethe particle.

Here, it is advantageous to measure not only the thermal stabilizationby means of fluorescence signal, but also a possible aggregation of theparticles, ligands and/or particle-ligand complexes by means of theinventive extinction measurement. This should make it possible to sortout, for example, ligands which lead to a thermal stabilization as wellas to an aggregation.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following, preferred embodiments of the present invention aredescribed in detail by making reference to the Figures:

FIG. 1 shows the mode of operation of the inventive measurement of thescattering of light by measuring the dilution of the light transmission;

FIG. 2 shows the direct measurement of scattered light with a fixedscattered light detection angle according to the prior art;

FIG. 3 shows the development of fluorescence signals and extinctionsignals by moving the samples relative to the optical system;

FIG. 4 shows an embodiment for evaluating the extinction measurement;

FIG. 5 shows an embodiment for evaluating the fluorescence measurement;

FIG. 6 shows an embodiment for measuring fluorescence and extinctionsimultaneously;

FIG. 7a shows an embodiment for the almost simultaneous measurement offluorescence ratio and extinction with drawn-in path of fluorescencerays;

FIG. 7b shows the embodiment of FIG. 7a , however, with drawn-in path ofextinction rays;

FIG. 8a shows a further embodiment for the simultaneous measurement offluorescence ratio and extinction with drawn-in path of fluorescencerays;

FIG. 8b shows the embodiment of FIG. 8a , however, with drawn-in path ofextinction rays;

FIG. 9 shows the reflectivity of silicon;

FIG. 10 shows a measurement example for simultaneously detecting theintramolecular unfolding by means of fluorescence and the intermolecularaggregation by means of extinction of an antibody;

FIG. 11 shows a measurement example of the increase in aggregation of anantibody dependent on the temperature in different buffers;

FIG. 12 shows a measurement example for the detection of proteinstability at different temperatures by chemical unfolding;

FIG. 13 shows an exemplary measurement for the demonstration of thedynamic range of the fluorescence optics when different proteinconcentrations between 50 mg/ml and 2 μg/ml are used;

FIG. 14 shows an exemplary measurement for the quality control ofproteins by forced degradation tests;

FIG. 15 shows exemplary measurement data for the buffer screening foroptimum storage conditions of an antibody;

FIG. 16 shows an exemplary absorption spectrum of a protein; and

FIG. 17 a, b shows a top view and cross-sectional view of an inventivetempering body.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 2 shows a usual method for measuring particles by means of a staticscattered light measurement in a fixed angle. The sample 13 to beexamined is a liquid with strongly scattering or strongly aggregatingparticles. The sample liquid is in a capillary 30, which is arranged ona surface 77. For an extinction measurement light 20 is irradiated fromthe top downwards through the capillary 30 into the sample liquid. Onepart of the irradiated light 20 is directly, i.e. substantially oppositeto the irradiation direction, reflected back as reflected light 22. Forthe measurement of scattered light 24 a scattered light detector 200 isin an angle Φ between irradiating ray of light 20 and sample and thusdirectly determines the light 24 which is scattered into the sample withstrongly scattering particles 13.

The disadvantages of the system may be summarized as follows. Thecontribution to the signal in the detector 200 is only generated by thescattering into a small angle range/range around the angle Φ. Due to themeasurement in a small angle range, the system is prone to undesiredmechanical movements, for example movements in the vertical direction.In certain positions of the capillary 30 reflections at the capillarywalls (for example ray 25) in the direction of the detector 200 arestronger than the light scattering at the particles to be examined Aperson skilled in the art of scattered light measurements knows that itis important to avoid reflections or reflecting surfaces 77 (for examplesilicon) since from there for example an undesired reflected ray 26 mayalso enter the detector 200. Said reflected ray 26, which enters thescattered light detector 200, leads to falsification of the measurementsignal, since for scattered light measurements only a very small anglerange around the angle Φ may be measured according to the conventionalteaching. Thus, the skilled person will construct a very complex opticalsystem in order to block all undesired scattered light, which, however,makes the optical system fragile and expensive. In particular, a skilledperson will avoid reflecting surfaces. Furthermore, the inclinedarrangement of the detector 200 impedes the integration in existingoptics with vertical path of rays.

FIG. 1 schematically shows the mode of operation of an inventivemeasurement. Preferably, according to the invention, the scatteredportion of light is not measured directly as in FIG. 2, but bymeasurement of the dilution of the light transmission, the so-calledextinction. In other words, the extinction light is light which is notscattered. Dependent on the configuration of the optics, light which isscattered less than ±10°, preferably less than ±8°, 7°, 6°, 5°, 4°, 3°,2°, 1° from the ray axis A of the irradiated light 20 is preferablyinterpreted as light which is not scattered. When having a highacceptance angle range, a high signal-noise-ratio may be reached, whenhaving a small range, the linearity is better at high concentrations.

Again, in this example, the sample to be examined is in a capillary 30which rests on a surface 77. The light of an arriving ray of light 20 isscattered by particles in the sample solution 13 partly under differentangles (cf. scattered light 24). The ray of the irradiating light isreflected at the surface 77 and returns as ray of light 22 opposite tothe irradiated ray of light 20. The intensity of the ray of light 20, 22which has been reflected at the surface 77 and thus twice ran throughthe sample volume 13, depends on the intensity of the light scatteringin the sample. The intensity of the reflected ray 22 is measured by adetector 100, whose acceptance range is collinear to the ray of light 20or the rays of light 20, 22. The wavelength of the arriving/irradiatedrays of light 20 and the reflected rays of light 22 is preferably chosensuch that the sample to be measured absorbs as little light as possiblein said range. Thus, it may be achieved that the dilution of the lightis predominantly effected by scattering (extinction) and not byabsorption. A further advantage of said inventive method is that rays 26which are reflected at the surface 77 do not disturb the measurement.

FIG. 17a shows the surface 77 of the inventive tempering body withseveral capillaries 30 arranged thereon in top view. The surface 77 hasa length L and width B, wherein the surface layer furthermore has adepth T, as can be seen from FIGS. 17a and 17b . Preferably, the lengthL is longer than the width B. Furthermore, it is preferred that for themeasurement the capillaries 30 extend along the width B of the surface77 and the capillaries are preferably longer than the width B so thatboth ends of the capillaries project over the surface 77. In order totemper the capillaries 30 via contact, it is preferred that thecapillaries directly rest on the surface 77 of the tempering element,i.e. are in direct contact to the surface 77. According to a furtherpreferred embodiment it may further be advantageous to configure atleast one region such that a portion of the capillary is not in directcontact to the surface while other region of the capillary are incontact with the surface. In particular, a region without direct contactis advantageous for optical measurements, as will be discussed in thefollowing.

According to a preferred embodiment a recess 90 may be provided in thesurface 77, for example in the form of a furrow, groove, micro groove or“ditch” 90 so that there is no direct contact of the capillary to thesurface 77 in the region of the groove 90. The groove 90 preferablyextends over at least a region of the tempering element on which thecapillaries rest during the measurement. The groove 90 is preferablyconfigured in the central region with respect to the width of thetempering element so that each capillary has no direct contact to thesurface 77 in a central measurement region 90. However, right and leftof said region 90 (with respect to the width of the tempering element)the capillary 30, is in direct contact to the surface in order to securea contact tempering.

The groove is preferably between 1-10 mm, more preferably between 2-8mm, further preferably between 3-7 mm, for example 5 mm, furtherpreferably has a width of approximately 3 mm (along the width B).According to the invention the inventive reflection of the light isproduced or measured preferably in said groove portion of thecapillaries.

Preferably the groove is approximately 10-30 μm deep. It is particularlypreferred that the groove 90 has a depth (see direction of depth T inFIG. 17b ) of more than half of the coherence length of the used lightin order to further suppress interference effects in the backscattering.Disturbing interference effects for example occur due to Newton's ringswhich may be suppressed or even avoided with an inventive groove.According to the invention, a laser light source or an LED may be usedas light source. LED light sources, as they are for example used in thepresent invention, typically have coherence wavelengths in the range ofapproximately 15 μm so that a depth of the groove of >7.5 μm ispreferred. It is particularly preferred that the depth is between 1.5times of half of the coherence wavelength and 10 times of half of thecoherence wavelength. Preferably, the upper limit of the depth is 5times of half of the coherence wavelength. In particular, according tothe invention, the groove should be only deep enough to suppressinterferences, in return, however, there should not be an air cushionbelow the capillary which is too big since in this case the desiredtemperature in the capillaries could be disturbed by the air cushion.Furthermore, the groove has the further preferred advantage that thesurface of the capillary 30 is not in direct contact with the surface 77so that scratching of the surface of the capillary 30 and scratching ofthe surface of the tempering body by the capillary may be suppressed oravoided in the measurement region (groove). In particular, according tothe invention, it may be avoided that the surface of the tempering bodyis scratched, whereas a possible scratching of the capillaries may betolerable since the capillaries are preferably used as disposablearticle. According to the invention, for example capillaries may be usedwhose material has a lower hardness than silicon.

In order to guarantee an more efficient reflection of light from thebottom of the groove 90, the groove is preferably etched into thesurface of the tempering element. Preferably, the tempering element hasa surface layer made of silicon so that the groove 90 is configureddirectly in the silicon layer. According to a preferred embodiment ofthe invention, the groove is etched into the silicon. Furthermore, thepreferred etching method has the advantage that the surface of thebottom of the groove is configured in a very smooth way so that thereflection behavior of said surface is still excellent. Preferably, thesurface of the bottom has an average roughness which is preferably inthe nanometer range, preferably <±10 nm, preferably <±5 nm, for example±1-2 nm.

According to a preferred embodiment, the groove may extend over asubstantial part of the surface so that for example all capillaries 30which have to be measured and rest on the surface 77 may be arrangedabove the groove 90. As illustrated in FIG. 17a , the groove 90 extendsalong the length L so that the capillaries 30 may be arrangedtransversely over the groove 90 (cf. FIG. 17b ). According to a furtherpreferred embodiment, the groove 90 does not extend over the completelength L so that preferably in the edge region 91 no groove isconfigured. This has, for example, the advantage that the silicon has aconstant thickness around the groove 90 and may thus be easier processed(for example cutting or sawing).

Preferably, silicon is used as surface of the tempering element.Preferably, pure (crystalline) silicon is used, as discussed in detailfurther below. Preferably, the inventive groove 90 is configured along apreferred crystallographic direction of the crystalline silicon,preferably along the [111] direction (Miller's direction indices).

For example, the groove also has the advantage that liquid which is atthe outside of the capillary does not reach the measurement region whichis preferably in the region of the groove. Since in the region of thegroove the distance between capillary and tempering body is larger thanoutside the region of the groove, it is favorable that the liquid at theoutside of the capillary stays outside the groove because of thecapillary forces.

Thus, it may happen, for example, that when the capillaries are filledsometimes droplets stick at the outside of the capillary. Said dropletsmay disturb when they reach the measurement region. However, thecapillary forces, which are greater the smaller the distance fromcapillary to tempering body is, hold said liquid outside the groove.Thus, it may, for example, be avoided that the liquid of the dropletsreach the measurement region in the groove.

FIGS. 3a ) to 3 c) show the development of the signals for an inventivefluorescence measurement and extinction measurement. Similar to FIG. 1,the sample to be examined is in a capillary. The sample to be examinedcontains scattering/aggregating particles (in the following referred toas sample 12) as well as fluorescent particles (in the followingreferred to as sample 15). In order to measure the sample, preferablythe detector is shifted above the capillary or the capillary is shiftedbelow the detector. Said shifting is conducted preferably transverselyto the longitudinal axis of the capillary. Alternatively, capillary aswell as detector may be shifted. However, preferably a relative movement80 between capillary 30 and detector 100 is supposed to happen during ameasurement.

Before a capillary 30 is reached by the irradiated rays of light for thefluorescence measurement and extinction measurement 20, 21, the detectordoes not measure a fluorescence light 23 (upper row; signal(fluorescence)) and no dilution in the reflected light 22 for theextinction measurement 22 (lower row; signal (extinction)).Correspondingly, a horizontal line is shown in the diagrams in FIG. 3 a.

During the (relative) movement 80 of the capillary 30 under thedetection region of the optical system, the measured fluorescenceintensity 23 increases and the intensity of the reflected ray 22decreases by refraction at the capillary and by scattering in the sample12 (cf. FIG. 3b ).

When the capillary with the sample leaves the detection region of theoptics, preferably signals are generated which correspond to the signalswhich are generated when driving into the detection range. This iscaused by the symmetrical arrangement of the optical system or thesymmetrical movement above the capillary. Thus, the direction 80 of themovement between samples and optical system is irrelevant.

According to the invention, a plurality of samples which are in aplurality of capillaries may be measured continuously after each other.The plurality of capillaries may be arranged preferably on a samplearray. That means by a preferably continuous movement of a sample arraya plurality of samples with high data density (data points per sampleper time unit) may be recorded. Thus, it is for example possible toobtain measurement frequencies up to more than 100 kHz. A furtheradvantage of said inventive method is the low adjustment effort of thesystem. Furthermore, capillaries 30 as format for a sample chamber makesimple filling possible by automatically filling the sample into thecapillary by capillary forces, which for example also makes it possibleto fill in highly viscous solutions. The capillaries preferably directlyrest on the surface 77 and have good heat contact.

FIG. 4a ) shows a cross-section through three different samples 11, 12,13 in capillaries 30. The first sample 11 does not scatter the light 20produced and emitted by a source so that the largest portion 22 of theirradiated light rays 20 is reflected back in the direction of theobjective lens and detector 100. The other two samples 12 and 13 scattera portion of the irradiating light 20 in different directions outsidethe acceptance angle of the detector 100. A larger part of theirradiating light is scattered through the sample 13 than through thesample 12, which is shown by the plurality of scattering arrows 24.

As already described with respect to FIG. 3, it is preferred that duringa measurement the samples are moved relatively to the optical system.FIG. 4b ) shows a typical course of the light intensity measured by thedetector 100 dependent on the horizontal position of the samples(extinction measurement). The measured intensity (brightness [I])depends on the one hand on the light diffraction at the capillary walls30 and on the other hand on the extinction in the sample. The lightdiffraction at the capillary walls may be assumed to be identical in agood approximation in different samples. Since the samples 12 and 13,however, scatter a larger portion of the irradiating light 20 than thesample 11, less light 22 is reflected back. Thus, the brightness(intensity) in samples 12 and 13 decreases to a greater extent.

FIG. 4c ) shows a possible course of the extinction dependent on theposition of the samples. The formula for calculating the extinctiongenerally is

${E(x)} = {- {\log_{10}\left( \frac{I(x)}{I_{0}(x)} \right)}}$

I0(x) may be a constant in the simplest case or the course of intensityin a capillary filled with water or the course of intensity when ameasurement is started before the extinction has started because oftemperature-induced formation of aggregation.

The desired measurement value “extinction” (FIG. 4d ) results fromintegration of the extinction course E(x). The integration limits arepreferably symmetrical around each capillary. In order to balancefluctuations of the brightness of the light source or the sensitivity ofthe detector, the detected extinction may be corrected by a referencevalue which is calculated by integration of the curve E(x) in a rangewithout capillary (cf. FIG. 4c : “reference surface”). Preferably, saidcorrection is carried out for each capillary individually with a regionwithout capillary directly next to said separate capillary. According tothe invention, this is for example possible since the sample, contraryto measurement methods from the prior art, is moved preferably relativeto the measurement system.

FIG. 5 exemplarily explains a possible processing of the measurementdata from FIG. 3 using the example of three samples with high 14,average 15 and low 16 fluorescence.

The intensity of the fluorescence light which is emitted by the samplesis shown in FIG. 5b dependent on the movement or the position of thedetector 100 to the capillary. In order to determine a “fluorescencevalue”, it is integrated over the value of the shifting 80 of thesamples relative to the optical system (cf. FIG. 5c ). The integrationlimits comprise preferably symmetrically one separate capillary. Theintegrated fluorescence intensity preferably corresponds to themeasurement value of the fluorescence of a sample which is to bedetermined. It is also possible to measure the fluorescence intensitiesat two or more different wavelengths. In this case the ratio of theintegrated fluorescence intensities is the measurement value“fluorescence ratio” which is to be determined.

FIG. 6 shows an exemplary configuration of an inventive system formeasuring fluorescence and extinction. Preferably, said measurement offluorescence and extinction may be conducted after each other, almostsimultaneously or simultaneously. A (first) light source 40, for examplean LED, generates light radiation 21 with a (first) wavelength 21, whichstimulates the emission of fluorescence radiation in the sample volume10. The stimulation filter 70 suppresses possible radiation of the lightsource 40 in wavelengths ranges which are not desired. A (second) lightsource 41 generates light radiation 20 in a (second) wavelength range inwhich the sample volume 10 has only little absorption. The light of bothlight sources 40, 41 is preferably collimated with the optical lenses60, 61 and combined to a collinear ray by a dichroic beam splitter.

The beam splitter 72 has a high reflectivity preferably in the (first)wavelength range 40. Furthermore, it is advantageous when the beamsplitter 72 comprises a high transmission in the wavelength range of thefluorescence emission of the sample. It is also further preferred thatthe beam splitter 72 comprises partly a transmission and partlyreflection in the wavelength range of the (second) light source 41. Saidrequirements are for example fulfilled by a dichroic beam splitter whenthe wavelength of 41 matches with the fluorescence emission of 10. Inthe more general case the beam splitter 72 is a trichroic beam splitter.

The beam splitter 72 reflects the light of the first and secondwavelengths 20, 21 to the sample 10 in the capillary 30. The objectivelens 62 focuses the irradiating light to the sample 10. The light of thefirst light source 40 generates in the sample 10 fluorescence radiationwhich is collimated by the lens 62. The light in the irradiating beam,which is generated by the second light source 41, arrives through thesample 10 and the capillary 30 at the surface 77, is reflected orbackreflected and runs a second time through the sample 10 and thecapillary 30.

The surface 77 is preferably made of a material having littlefluorescence on its own and having high reflectivity in the wavelengthrange of the second source 41 in order to measure extinction. Thereflected radiation is again collimated by the lens 62. Particles whichare possibly present in the sample volume scatter the irradiating lightso that only a smaller part of the originally irradiated light isabsorbed by the objective lens 62. Thus, the intensity of the lightwhich is reflected back to the lens 62 substantially depends on theconcentration and dimension of the particles and thus on the extinctionof the sample.

The filter 73 preferably suppresses the fluorescence excitation light ofthe (first) light source 40. The detector 53 measures, preferably in awavelength-selective manner, the intensity of the ray of light comingfrom the sample. Preferably, the light of the second wavelength, i.e.the light which passes through the sample and is reflected back, as wellas the fluorescence light, i.e. light of the fluorescence emission ofthe sample, are measured by the detector 53. Furthermore,wavelength-selectively means that the intensities at the differentwavelengths may be determined preferably separately from each other.

FIG. 7a ) shows a further inventive embodiment of the system withdrawn-in path of rays during fluorescence measurement. However, contraryto FIG. 6, the system has (at least) two detectors. Both detectors 50,51 serve for the measurement of fluorescence intensity at two differentwavelengths. For example, if 330 nm and 350 nm are chosen as detectedwavelengths, the ratio of both signals provides information regardingthe structure of macromolecules which are present in the sample volume10.

In said embodiment the wavelength of the (second) light source 41 forthe extinction measurement is in the range of the fluorescence emissionof the sample 10. Thus, during the measurement of fluorescence the lightsource 41 should be switched off. A sequential or almost simultaneousmeasurement is generated due to a fast and alternating measurement ofextinction and fluorescence. The time which passes between two datapoints of a measurement type has to be so short that the differencebetween two measured values is less than the measurement uncertainty ofa measured value. Example: extinction of a highly concentrated samplechanges at temperatures over 80° C. with approx. 0.2 mAU/s (milliabsorption units/second). The measurement uncertainty with respect tothe extinction is for example approx. 0.2 mAU. Correspondingly,extinction is measured preferably at least 1× per second andfluorescence also at least 1× per second. In said embodiment thebandpass 73 transmits one part of the fluorescence radiation 23, forexample in the range of 320 nm to 360 nm. The beam splitter 74 separatesthe fluorescence radiation into two rays with wavelength ranges of forexample 320 nm-340 nm and 340 nm-360 nm. The rays are bundled with theconcentrator lenses 63, 64 onto both detectors 50, 51. The quotient ofboth measurement signals is the measured value to be determined.

FIG. 7b ) shows the exemplary embodiment of the system of FIG. 7a ),however, with drawn-in path of rays during the extinction measurement.The second light source 41 emits light radiation 20 which twice runsthrough the sample 10 after reflection at the base plate 77 and runsupwards as ray of light 22. In the sample 10 the intensity of the ray oflight is diluted by scattering from the detection range. The wavelengthof the (second) light source 41 preferably is in the transmission rangeof the filter 73. Depending on the wavelength of the light source 41 thelight then disperses to both detectors 50, 51. Preferably, thewavelength is at approx. 350 nm so that the largest part of the light ismeasured by one single detector.

FIG. 8a ) shows an exemplary embodiment of the system of FIG. 6 which isexpanded by an additional detection branch, compared with the embodimentin FIG. 7. The path of rays for the fluorescence, which corresponds tothe path of rays in FIG. 7a , is drawn in. The additional dichroic beamsplitter 75 is transparent in the wavelength range which is measured bythe detectors 50, 51.

FIG. 8b ) shows the system of FIG. 8a ) with drawn-in path of rays forthe extinction measurement. Compared to the system of FIG. 7, thewavelength range of the light source 41 lies outside the wavelengthrange which is measured by the detectors 50, 51, for example 380 nm.Thus, the light source 41 may be switched on during the measurement andit does not have to be switched between the measurement typesfluorescence and extinction. The beam splitter 72 is partly transparentin the wavelength range of source 41. Ideally, thetransmission/reflection ratio is 1:1.

The beam splitter 75 directs the light from the (second) light source 41to the detector 52 after it has been diluted by extinction in the sample10. The bandpass 76 preferably reduces the share in fluorescence lightto the detector 52.

Due to said embodiment with three detectors 51, 52, 53 extinction andfluorescence ratio may be measured continuously and simultaneously.Furthermore, the sensitivity of the detector 52 may individually beadapted to the intensity of the radiation from the light source 41. Thesensitivity of the detector may be adjusted for example significantlyhigher for a noise-reduced measurement of the extinction. In anadvantageous embodiment the signals of the detectors are digitalized bya 24 bit analog digital converter (ADC) which may read in simultaneouslyall three detector channels, for example with a rate of 4 kHz.

FIG. 9 is a diagram in which the reflectivity is shown dependent on thewavelength for silicon. In particular, FIG. 9 shows the goodreflectivity of silicon in the UV range, which is particularly preferredso that the light intensity reflected to the detector is as high aspossible during the extinction measurement. In particular, a high lightintensity enables a measurement having little noise. Furtheradvantageous properties of silicon are that the used wavelengths havealmost no fluorescence themselves, that they may be mechanicallymanufactured easily and that they have high chemical resistance.

FIG. 10 exemplarily shows a measurement with the inventive systemdescribed in FIG. 6. The unfolding of proteins dependent on thetemperature as well as the aggregation of an antibody dependent on thetemperature is shown. In the shown example the unfolding of one of thesub-units of the antibody starts already at 60° C., which ischaracterized by a characteristic change in the fluorescence ratiobetween the emission at 350 and 330 nm. An increase of the aggregationand thus in the extinction, is observed only from 73° C. onwards, whichsuggests that an unfolding of the thermally more instable protein domaindoes not contribute to the aggregation of the antibody.

FIG. 11 exemplarily shows the measurement of extinction of an antibody(rituximab) having a substance concentration of 1 mg/ml in 25 mM acetatebuffer at different pH values between pH4 and pH6. 10 μl of the solutionwas heated in each capillary from 50 to 95° C. with a heating rate of 1°C./min. An increase in the extinction can be observed at increasedtemperatures >72° C., which may be explained by aggregation. The extentof the extinction increase depends on the pH value of the solution,wherein lower pH values counteract the temperature-induced aggregation.This is characterized on the one hand by a late start of the extinctionincrease (“aggregation-onset temperature”) and on the other hand by atotal lower maximum extinction.

FIG. 12 exemplarily shows the analysis of the chemical stability of theprotein lysozyme in 10 mM citrate buffer pH4 at differentguanidine-hydrochloride concentrations. 1 mg/ml lysozyme was preparedwith increasing guanidine-chloride concentration in 48 solutions and 10μl of each solution was filled into capillaries, said capillariesarranged and fixed on the capillary array and subsequently eachcapillary scanned at 20° C., 30° C. and 40° C. Subsequently, theobtained fluorescence ratios are set in relation to increasing guanidineconcentrations. In particular, the fluorescence ratio shows in allsamples a sigmoidal increase when the guanidine concentration increases,which is directly proportional to the portion of unfolded protein. Whenthe temperature increases, the protein more and more destabilizes, whichis characterized by a shifting of the data points to lower guanidineconcentrations.

FIG. 13 shows the exemplary measurement of the protein streptavidin inPBS, pH 7.3, at different substance concentrations of 50 mg/ml to 7μg/ml. The capillary scan illustrates the different fluorescenceintensities in the capillaries. The upper diagram shows the capillaryscan at the beginning of the measurement of the thermal unfolding. Allconcentrations are measured as duplicates. The height of the peakscorresponds to the fluorescence intensity in the capillaries at anemission wavelength of 350 nm. The decrease of the fluorescence at highstreptavidin concentration can be explained by the inner filter effect,which is generated by the strong absorption of the excitation light andreduced intrusion depth caused thereby (thus lower fluorescence). Thelower diagram shows the course of the temperature of the fluorescenceratio at 350 nm and 330 nm. Said unfolding curves show that unfoldingprofiles have been recorded for all concentrations. At allconcentrations a clear unfolding process may be recognized. The meltingtransition is shifted to higher temperatures at high streptavidinconcentrations, which is due to an intramolecular stabilization of theprotein.

FIG. 14 shows exemplary data of a forced degradation test for theprotein MEK1-kinase. A solution with a concentration of 1 mg/ml in 50 mMhepes pH 7.4, 150 mM NaCl and 2 mMDTT was prepared and divided into 5aliquots à 50 μl. While an aliquot was stored at 4° C. and served asreference, the remaining aliquots were exposed to differentconditions—incubation at increased temperature, freezing-unfreezingcycles, strong stirring. Subsequently, all samples were filled intocapillaries, placed on the capillary array and pressed on, and thethermal unfolding detected at a heating rate of 1° C./min from 25° C. to90° C. via the fluorescence. The upper diagram shows the unfoldingcurves of the samples. Depending on the previous treatment, the startinglevels of the unfolding curves are different, which suggests differentshares of already unfolded protein. The lower diagram shows aquantification of the share of unfolded protein in %, wherein the sampleof the 4° C. incubation unfolds as 0% and the sample after 15 minutes ofincubation at 60° C. was used as reference.

FIG. 15 shows exemplary data of a buffer screening for theidentification of optimum conditions for the storage of antibodies. Amonoclonal antibody was stored at a concentration of 5 mg/ml in acetatebuffer with different pH values as well as in the absence and thepresence of 130 mM NaCl. 10 μl of each antibody solution wassubsequently filled into glass capillaries and the temperature-dependentunfolding of proteins was measured via the change in fluorescence andthe temperature-dependent aggregation was measured via the increase ofextinction at a heating rate of 1° C./min FIGS. 15a ) and b) show thetemperature-dependent increase in the aggregation. In the shown case thetotal aggregation increases with increasing pH value, which ischaracterized by higher amplitudes in the aggregation signal. Theaddition of physiological salt concentrations leads to a furtherincrease in the aggregation at all pH values (b). Figs. c) and d)exemplarily show the determination of the aggregation-onset temperature,which corresponds to the lowest temperature at which a significantincrease of the extinction in relation to the base line is observed.Fig. d) exemplarily shows the different dependency of the aggregationtemperature on the pH value and the salt concentration. Figs. e) and f)show fluorescence data which are recorded, according to the invention,simultaneously with the aggregation data shown in FIGS. 15a ) and b).With an increasing pH value of the solution, the antibody shows higherthermal stability. Furthermore, NaCl has negative effects on thermalstability, which can be recognized by means of an unfolding of theproteins at lower temperatures. By comparable experiments conditions maybe detected under which the thermal stability of a protein, for examplean antibody, is maximal and the aggregation is minimal.

FIG. 16 shows an exemplary absorption spectrum of a protein.

The invention also comprises the accurate or exact expressions,features, numeric values or ranges etc when said expressions, features,numeric values or ranges are before or subsequently named with termslike “approximately, about, substantially, generally, at least” etc(i.e. “approximately 3 should also comprise “3” or “substantiallyradial” should also comprise “radial”).

LIST OF REFERENCE SIGNS

-   10: sample-   11: sample without scattering/aggregating particle-   12: sample with some scattering/aggregating particles-   13: sample with strongly scattering/aggregating particles-   14: sample with many fluorescent particles-   15: sample with some fluorescent particles-   16: sample with few fluorescent particles-   20: irradiated light for the extinction measurement-   21: excitation light for fluorescence-   22: reflected light-   23: emission light fluorescence-   24: scattered light in specific scattering angle Q-   25: “undesired” scattered light-   26: “undesired” reflected scattered light-   30: capillary-   40: light source for fluorescence excitation-   41: light source for extinction-   50: detector 1 (fluorescence and extinction)-   51: detector 2 (fluorescence and extinction)-   52: detector 3 (extinction)-   53: detection system-   60: collimator lens for 40-   61: collimator lens for 41-   62: objective lens-   63: concentrator lens for 50-   64: concentrator lens for 51-   65: concentrator lens for 52-   70: excitation filter for 40-   71: beam splitter for the combination of 40+41-   72: beam splitter for separating excitation and fluorescence-   73: fluorescence emission filter-   74: beam splitter for separating fluorescence-   75: beam splitter for separating fluorescence and extinction-   76: extinction filter-   77: reflecting, non-fluorescent surface, for example silicon surface-   80: running directions of the capillary array-   90: groove, furrow, ditch, recess-   91: edge region-   100: inventive alternative for detection-   200: detection optics for scattered light according to the prior art

The invention claimed is:
 1. A method for optically measuring thethermal stability of viruses in a liquid sample, which is in a samplecontainer, wherein the method comprises the following steps: irradiatingthe sample with light of at least a first wavelength, to fluorescentlyexcite the viruses, irradiating the sample with light of at least asecond wavelength to examine the scattering of the viruses, measuringthe fluorescence light emitted by the sample; and measuring theextinction light at the second wavelength, wherein the irradiated lightof the second wavelength runs through the sample container, is reflectedback, runs again through the sample container in opposite direction andexits as extinction light, wherein the stability of the viruses ismeasured on the basis of the measured fluorescence light and theaggregation on the basis of the measured extinction light, wherein thesample container is shifted during a measuring period relatively to theirradiated light of the first and/or second wavelength and/or to thedetector and is driven back and forth several times; and wherein aplurality of sample containers or a plurality of capillaries are scannedby said relative movement.
 2. The method according to claim 1, whereinthe sample container is tempered and the stability measurements arepreferably performed at different temperatures.
 3. The method accordingto claim 2, wherein the sample container rests on a tempering elementand is tempered by a contact.
 4. The method according to claim 3,wherein the tempering element further reflects back the irradiated lightof the second wavelength, again runs through the sample container inopposite direction and exits as extinction light.
 5. The methodaccording to claim 3, wherein the tempering element is made of amaterial i) which has little autofluorescence <1%, and/or ii) which hasa high reflectivity >30% in the wavelength range of the secondwavelength and preferably comprises silicon or consists of pure silicon.6. The method according to claim 3, wherein at the surface of thetempering element at least one groove is configured, the samplecontainer is arranged above the groove and the irradiated light of thesecond wavelength is reflected back from the bottom of the groove. 7.The method according to claim 6, wherein the groove has a width between1-10 mm and a depth of more than half of the coherence length of thelight of the second wavelength.
 8. The method according to claim 2,wherein during a measuring period the temperature of the samples ischanged, preferably increased to determine the thermal stability.
 9. Themethod according to claim 2, wherein the temperature is continuouslyincreased by a rate of at least 0.5° C. per minute, preferably 1° C. perminute.
 10. The method according to claim 1, wherein the thermalstability of the viruses is determined by identification of inflectionpoints, preferably by analysis of the first or second derivation of theraw data or by other mathematical processes.
 11. The method according toclaim 10, wherein the inflection points are used to determine if thevirus contain RNA or DNA.
 12. The method according to claim 1, whereinthe fluorescence light and the extinction light are measured with acommon optical system.
 13. The method according to claim 1, wherein theirradiation of the sample i) is not conducted simultaneously with thefirst and second wavelengths; or ii) the irradiation with the secondwavelength is conducted continuously, whereas the irradiation with thefirst wavelength is conducted intermittently, preferably periodically.14. The method according to claim 1, wherein the fluorescence light andthe extinction light are measured simultaneously.
 15. The methodaccording to claim 1, wherein i) the extinction light and thefluorescence light are measured by a common detector; ii) the extinctionlight is measured by a first detector and/or a second detector andfluorescence light of a first fluorescence wavelength is measured by thefirst detector and fluorescence light of a second fluorescencewavelength is measured by the second detector; or iii) the extinctionlight is measured by a first detector, fluorescence light of a firstfluorescence wavelength is measured by a second detector andfluorescence light of a second fluorescence wavelength is measured by athird detector.
 16. The method according to claim 1, wherein the samplecontainer is a capillary.
 17. The method according to claim 1, whereini) a fluorescence value is determined by integrating the intensity ofthe fluorescence light via the shifting and/or ii) an extinction valueis determined by integrating the intensity of the extinction light viathe shifting.
 18. The method according to claim 1, wherein during ameasuring period a plurality of sample containers and/or the opticalsystem are continuously driven back and forth several times and themeasurements of the fluorescence light and/or the extinction light areconducted during the movement.
 19. The method according to claim 1,wherein the second wavelength is chosen such that less than 1%, 0.1%,0.05% is absorbed by the sample or the particles in the sample.
 20. Themethod according to claim 1, wherein the light of the first wavelengthand the light of the second wavelength are united to a collinear raywhich is irradiated into the sample container.
 21. The method accordingto claim 1, wherein the extinction light of the second wavelength, whichis reflected back and leaves the sample container in the oppositedirection to the irradiation direction, deviates from the irradiationdirection 5° at most, preferably less than 2°, further preferred lessthan 1°.
 22. An apparatus for the optical measurement of the thermalstability of viruses in a liquid sample which is located in a samplecontainer, in particular according to claim 1, wherein the apparatuscomprises: a first light source for irradiating light of a firstwavelength into the sample container to fluorescently excite theparticles to be examined, a second light source for irradiating light ofa second wavelength into the sample container to measure the scatteringof the particles, a first detector for measuring the excitedfluorescence light which is radiated from the sample, a second detectorfor measuring extinction light at the second wavelength wherein theirradiated light of the second wavelength runs through the samplecontainer, is reflected back, runs again through the sample container inthe opposite direction and exits as extinction light and an evaluationmeans which determines the bonding based on the measured fluorescencelight and based on the measured extinction light.
 23. The apparatusaccording to claim 22 comprising a tempering element with a reflectingsurface at which the irradiated light of the second wavelength isreflected back, and wherein the apparatus is preferably configured toarrange at least one sample container on the surface for measurementpurposes.
 24. The apparatus according to claim 22, wherein the at leastone sample container is a capillary.
 25. The apparatus according toclaim 22, wherein the reflective surface consists of silicon, preferablyof crystalline silicon.
 26. The apparatus according to claim 22, whereinat least one groove is configured at the surface of the temperingelement, the sample container is arranged above the groove and theirradiated light of the second wavelength is reflected back from thebottom of the groove.
 27. The apparatus according to claim 26, whereinthe groove has a width between 1-10 mm and a depth of more than half ofthe coherence length of the light of the second wavelength.